To make a success of ADCs for cancer therapy, there are three points to overcome. First, ADCs gathered in solid tumor parenchyma based on the EPR effect should tether cancer antigens as receptors on the surface of cancer cells, even under static, stagnant conditions. Second, ADCs with payloads that exhibit their activity in the cytoplasm or the nucleus should be internalized into cancer cells across the plasma membrane via RME. Third, released payloads should escape from endosomes or lysosomes to the cytoplasm.

This first problem is a relatively challenging task. It is thought that the FSS at static, stagnant regions in solid tumor parenchyma is probably almost close to 0 Pa, or very little, whereas the cerebrospinal fluid (CSF) flows based on the glymphatic system in the brain parenchyma without the lymphatic system. Increasing the collision frequency and attachment level between ADCs and cancer antigens in solid tumor parenchyma can be accomplished by (a) increasing the number of ADC molecules, (b) increasing the ADC molecular size, or (c) rendering solid tumor parenchyma leakier.

2.7.2. Approaches That Increase the ADC Molecular Size to Up the Probability of Collision

(b) mAb-loaded nanoparticles containing payloads are a type of PEG engagerEGFR methodology. The size of the designed compounds should be contained in endosomes to deliver payloads with the activity in the cytosol or the nucleus via RME. Endosomes induced from clathrin-dependent endocytosis are 85–150 nm in diameter. However, strictly speaking, this strategy does not use orthodox ADCs, due to the use of nanoparticles. The interaction of the nanodelivery methodology to RME machinery and the endosomal–lysosomal system and that of the ADC delivery methodology to RME machinery and the endosomal–lysosomal system are different from the point of view of structural pharmaceutical science, resulting in an alteration of the half-life, RME mechanisms, and payload release. Although mAb-loaded nanoparticles containing payloads effectively tether cancer antigens and are transported into cancer cells via RME, they raise the manufacturing cost. Nevertheless, mAb-loaded nanoparticles containing payloads are promising anti-cancer agents.

PEGylation, covalently attaching polyethylene glycol (PEG) chains to peptides, proteins, or nanoparticles, is expected to improve water solubility, non-immunization by decreasing macrophage clearance, non-filtration by increasing the molecular mass, and enzymatic non-degradation by interfering with enzymes [86]. Compounds with a diameter of less than 6–8 nm are subject to filtration and excretion by kidneys from the blood stream into urine [87]. Compounds of approximately 500 Da to approximately 1500 Da, such as glucuronic acid conjugates, are subject to secretion by the liver into bile over biliary epithelial cells. Thus, ADCs are not supposed to be filtered and excreted by the kidneys and liver. However, ADCs can be avoided through ingestion by the reticuloendothelial system, which comprises a network of cells and tissues such as the blood, spleen, bone marrow, liver, and lymph nodes. Macrophages, Kupffer cells in the liver, and microglial cells in the brain play such an ingestive role. Phagocytosis, a type of endocytosis, by phagocytes such as macrophages forms phagosomes (1–3 μm in diameter) [88,89]. Macrophages exhibit the highest attachment to particles with a longest dimension of 2–3 μm, which is the same as the size of common bacteria [90]. Owing to such phagosomes, bystander ADCs might happen to be phagocytosed together with substances unrelated to them, even though ADCs are not activated by binding to their antigens. For accidentally phagocytosed ADCs, so as not to become antigens, human or humanized mAbs should be used in ADCs. Nonetheless, the internalization of ADCs binding FcγRs into phagocytes or antigen-presenting cells via RME might be avoided by appropriate PEGylation. When too many PEGs are introduced into ADCs, such PEG chains might inhibit the binding of ADCs to cancer antigens on cancer cells. The Flory radiuses of PEG100, PEG200, PEG300, and PEG400 are approximately 5 nm, 8 nm, 11 nm, and 13 nm, respectively, where PEGn would be n ethylene glycol repeats [91]. The radius is half of the diameter. PEG200 is almost as large as an IgG protein. Compared to nanoparticles, sizing up ADCs by PEGylation might be restricted due to molecular size, although a longer half-life and less immunogenicity were established [92]. Random PEGylation led to a loss of the biological potency of ADCs [93].Albumin (approximately 7 nm in diameter [94], 65–70 kDa) is the most abundant circulating protein in plasma, accounts for 55–60% of all plasma proteins [95], and is used as a carrier for drug delivery [96]. Thus, ADCs modified to enhance their ability to bind albumin in serum can increase the ADC molecular size by forming complexes. In fact, an IgG–albumin complex through disulfide linking at the hinge region between the Fab and the Fc fragments of IgG in a 1:1 ratio was formed in normal human serum [97]. Such naturally volume-enlarged IgG–albumin complexes (approximately 21.2 nm in diameter, based on summation) gathered in solid tumor parenchyma based on the EPR effect might enhance endocytosis. Human serum albumin (Figure 7 and Figure 8) has only one free cysteine residue (Cys34) and 34 cysteine residues that form 17 intramolecular disulfide bonds [98]. Moreover, 70–80% of all serum albumins have the free sulfhydryl group of Cys34 as a reductive from [99]. Intravenously administered ADCs with PEG, at the tip of which the free sulfhydryl group is introduced, might more easily form heterodimers such as a PEGylated mAb–albumin complex (more than 21.2 nm in diameter) at albumin Cys34 due to the predominant population of Cys34-free albumins in serum among all serum proteins; in addition, homodimers such as PEGylated mAb–PEGylated mAb probably formed in drug preparation. Oxidative stress is involved in the pathophysiology of all cancers. It is considered that serum is exposed to oxidative stress through the endothelial fenestrations at neovasculatures [100], which would enhance disulfide bond formation. Although 2-iodoacetamide moiety can react irreversibly with the sulfhydryl group, it is uncertain which sulfhydryl groups react in the living body. Thus, reversible disulfide linking is safe in pharmacological treatment. The disulfide linking between the mAb–albumin complex would be cleaved by the disulfide bond reduction in endosomes, just as PE38 was cleaved from Moxetumomab pasudotox-tdfk in endosomes [26]. The anti-HER2 nanobody 11A4 fused to an albumin-binding domain (ABD) at their C-terminus demonstrated the internalization into cells in in vitro assay, irrespective of albumin presence, using HER2-expressing cells. 11A4-ABD-maleimide-auristatin F showed a greater anti-tumor efficacy after a single-dose administration in in vivo assay using HER2-positive NCI-N87 xenograft-bearing mice, compared to 11A4-maleimide-auristatin F without ABD [43]. It was suggested that albumin was relevant to the EPR effect [43]. 2.7.3. Approaches That Render Solid Tumor Parenchyma Leakier(c) Solid tumors consist of the parenchyma and stroma. The systemic circulation and solid tumor stroma are virtually connected through a leaky vasculature. However, it is difficult for drugs to enter deeply into the inside of solid tumor parenchyma, because cancer cells are densely arranged there and the high pressure there prohibits drugs from advancing into the tumor. Moreover, it was thought that ADCs gathered in solid tumor parenchyma through a leaky vasculature based on the EPR effect were compelled to continue to remain due to little flow under static conditions (approximately 0 Pa), compared to the blood stream. If solid tumor parenchyma is leakier, ADCs could go through newly formed gaps along flows and could be endocytosed under low FSS. ADCs with IR700 can resolve this problem. IR700 is a dye that is activated by near-infrared (NIR) light (approximately 690 nm) and, consequently, injured the cell membrane. NIR light from the outside, peaking at 689 nm, can penetrate the living body to several centimeter depths from the surface without harmful side effects such as alteration of the immune system, unlike ultraviolet light. Thus, photodynamic therapy using IR700 will be a promising method for the treatment of solid cancers. Anti-HER1 panitumumab ADC possessing IR700 with a DAR of approximately 3 did not demonstrate tumor shrinkage after NIR light irradiation in in vivo assay using a tumor-xenografted mouse model bearing a 3T3/HER2 (HER1-negative) tumor in its dorsum, but it demonstrated tumor shrinkage after NIR light irradiation in in vivo assay using a tumor-xenografted mouse model bearing an A431 (HER1-positive) tumor in its dorsum [101]. When ADC–cancer antigen complexes are irradiated, they release their hydrophilic silanol units from IR700. As a result, such hydrophobic complexes are aggregated and make holes at the plasma membrane of cancer cells (Figure 9) [102]. With ADCs with IR700-induced cell rupture based on the membrane disruption, solid tumor parenchyma would be leakier than before. As a next strategy, the internal cytotoxicity by ADCs via RME could be effective. In the case of apoptosis, BAX/BAK oligomerization induces apoptotic pore formation at the mitochondrial outer membrane known as mitochondrial outer membrane permeabilization (MOMP), through which cytochrome c release into the cytoplasm is carried out [103]. Similarly, complexes of HER1 and ADC possessing IR700 without their hydrophilic silanol units might induce cytotoxic pore formation by their aggregation due to hydrophobic bonds or π–π stacking interactions. Eventually, alternative ADCs with payloads that show their activity in the cytoplasm or the nucleus could enter leakier solid tumor parenchyma.